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Search: Inhibition[Title] AND malic[Title] AND enzyme[Title] AND 1[Title] AND disrupts[Title] AND cellular[Title] AND metabolism[Title] AND leads[Title] AND vulnerability[Title] AND cancer[Title] AND cells[Title] AND glucose-restricted[Title] AND conditions[Title]

Abstract

Malicenzyme1 (ME1) regulates one of the main pathways that provide nicotinamide adenine dinucleotide phosphate (NADPH), which is essential for cancer cell growth through maintenance of redox balance and biosynthesis processes in the cytoplasm. In this study, we found that ME1 inhibition disrupted metabolism in cancercells and inhibited cancer cell growth by inducing senescence or apoptosis. In glucose-restricted culture conditions, cancercells increased ME1 expression, and tracer experiments with labelled glutamine revealed that the flux of ME1-derived pyruvate to citrate was enhanced. In addition, cancercells showed higher sensitivity to ME1 depletion in glucose-restrictedconditions compared to normal culture conditions. These results suggest that in a low-glucose environment, where glycolysis and the pentose phosphate pathway (PPP) is attenuated, cancercells become dependent on ME1 for the supply of NADPH and pyruvate. Our data demonstrate that ME1 is a promising target for cancer treatment, and a strategy using ME1 inhibitors combined with inhibition of glycolysis, PPP or redox balance regulators may provide an effective therapeutic option.

ME1 knockdown induces cellular senescence and suppresses cell growth. (a,b) HCT116 cells were reversely transfected with control, ME1, or ME2 siRNAs and 1000 cells were seeded on six-well plates. Then, cells were cultured for 11 days for colony formation assay. Colonies were fixed and stained with Crystal violet, and colony area of each well was quantified by GelCount and relative total area is shown as a bar graph (n=3, mean with s.d.). (c) Light microscopy images of colonies of HCT116 cells transfected with si control and si ME1-1. (d) PC3 cells were transfected with ME1 siRNAs and viability was determined 96 h after transfection. Relative cell proliferation is shown as a bar graph (n=3, mean with s.d.). (e) PC3 cells were transfected with ME1 siRNAs and cultured for 96 h. Cell size forward scatter (FSC) was detected by FACS analysis. (f) PC3 cells were transfected with ME1 siRNAs for 9 days and β-galactosidase was then stained. Cell (outer white line) and its nucleus (inner white line) are shown in the upper photograph. β-galactosidase was stained in blue and is shown in the lower photograph. (g) β-galactosidase-positive cells were manually counted and the percentage of stain-positive (SA-b-Gal positive) cells are shown in the bar graph. FACS, fluorescence-activated cell sorting.

Glucose depletion increases ME1-dependent glutamine metabolism and reduces expression of NADPH-producing enzymes other than ME1 in HCT116 cells. (a) HCT116 cells were cultured in glucose-depleted or -limited media (2, 1, 0.5 or 0.2 g/l glucose or glucose-free) for 72 h. Cell viability was determined at 24, 48, and 72 h using CellTiter Glo. Relative cell growth, normalised by cell viability at Day 0, is shown in the line graph (n=3, t-test; *P<0.01). (b) HCT116 cells were cultured in media with or without glucose for 24 h and labelled with [U-13C, U-15N] l-glutamine isotope for an additional 24 h. Then, isotope profiling analysis was conducted. Percentage of the pool of metabolites and isotopes is shown in the bar graph (2 g/l glucose in black and glucose-free in white; n=3, mean with s.d.). (c) HCT116 cells were cultured in media with 2, 1, 0.5 or 0.2 g/l glucose for 72 h and mRNA expression levels of NADPH-producing enzymes at 0, 24, 48 and 72 h were determined by TaqMan PCR and are shown in the line graph (n=3).

ME1 knockdown by shRNA induces metabolic reprograming and synergistically suppresses cancer cell growth in glucose-depleted conditions. HCT116 cell clones (sh ME1-1 and sh ME1-2), which are stably expressing tetracycline-inducible ME1 shRNA, were established. (a) Control shRNA, sh ME1-1 or sh ME1-2 clones were cultured in the media with or without 2 μg/ml doxycycline for 48 h, and ME1 expression was detected by TaqMan PCR. ME1 expression was normalised by GAPDH expression and relative ME1 expression is shown in the bar graph (n=3). (b) Cell numbers were determined 72 h with or without 2 μg/ml doxycycline. Relative cell number is shown in the bar graph (n=3, mean with s.d., t-test; *P<0.01) (c) Control shRNA, sh ME1-1 or sh ME1-2 were cultured in the media with or without 2 μg/ml doxycycline for 96 h and, d-glucose and l-lactate concentration in the media were measured. Relative glucose or lactate concentration are shown in the bar graph (n=3, mean with s.d., t-test; *P<0.01). (d,e) [U-13C, U-15N] l-glutamine isotope profiling analysis was conducted in sh ME1-1 and sh ME1-2 clones. Clones were cultured in the media with 2 g/l glucose with or without doxycycline for 72 h and labelled for additional 24 h in the glucose-depleted media with or without doxycycline. Then, metabolites and isotopes were measured. Peak area of labelled metabolites are shown in the dot graphs (n=3, t-test; *P<0.01; #P<0.05). (f) Three clones were cultured for 24 h in 2 g/l medium with or without doxycycline, and additionally cultured for 72 h in medium with 2 or 0.5 g/l glucose with or without doxycycline. Then, cell numbers were determined by cell counter (n=3, mean with s.d.; t-test; *P<0.01).